Microorganisms play a critical role for producing industrial products. Thus, it has been an object to conduct production more efficiently and at a lower cost. These methods of solving this object were to select a strain showing a higher productivity, and to examine the culture conditions such as medium composition for culturing microorganisms, culture temperature, etc. Under recent development of molecular genetics, as one alternative for such strain, a technique of specifying an excellent gene from a conventional strain, and utilizing the gene for transforming a strain can be exemplified. Conventionally, in yeasts that have been used to produce useful foods, transformation has been widely performed to achieve more effective production.
As one transformation to achieve effective production, a transformation for enhancing flocculation property of the cells can be exemplified. Alcohol production by fermentation method is conducted by the use of a technique of a batch fermentation or continuous fermentation, using a strain having a particularly high alcohol productivity among Saccharomyces cerevisiae, which is a fermentation yeast. Conventional batch fermentation method comprises adding molasses etc. to alcohol fermentation yeast as raw material, and culturing it under a certain condition to generate alcohol. The generated alcohol is recovered by distillation by heating the culture solution. However, yeasts remaining in the culture solution are killed by heating. Therefore, it is necessary to supplement yeast solution to continue alcohol production. Such process is inefficient and involves a high cost. When flocculent yeasts are used, it is possible to recover alcohol in the supernatant while allowing the solution to stand still and to add a new fermentation solution to the precipitated flocculent yeast to conduct again the alcohol production. Therefore, flocculent yeasts were awaited in the alcohol production by batch fermentation.
As a technique of transformation to confer flocculation property, the following can be exemplified:    A practical flocculent alcohol-fermenting yeast produced by introducing a flocculation gene expression cassette which is a foreign DNA into any marker gene region on the chromosome of alcohol-fermenting yeast, and a method of breeding the same (Patent Document 1); a method for producing yeast having an enhanced flocculation property comprising obtaining a DNA encoding a protein domain associated with the flocculation property of the flocculation gene of yeast, and introducing the DNA into beer yeast (Patent Document 2); and establishment of flocculent yeast for fuel ethanol production (Nonpatent Document 1). These transformed yeasts that have been already reported are produced by introducing flocculation gene derived from Saccharomyces cerevisiae into alcohol-producing yeast belonging to Saccharomyces cerevisiae. On the other hand, transformed yeast wherein flocculation gene derived from Saccharomyces cerevisiae has been introduced into yeast belonging to a genus other than Saccharomyces cerevisiae has not been reported so far, and it was not known at all whether flocculation gene derived from Saccharomyces cerevisiae is associated with the control of flocculation property in yeast other than Saccharomyces cerevisiae or not.
The whole genome has been analyzed in Saccharomyces cerevisiae, and the flocculation genes have been specified. The set of FLO genes associated with flocculation property include FLO1 gene present on the 1st chromosome (Nonpatent document 2); FLO5 gene present on the 8th chromosome (Nonpatent document 3); FLO8 gene present on the 5th chromosome (Nonpatent document 4); FLO9 gene present on the 1st chromosome (Nonpatent document 5); FLO10 gene present on the 11th chromosome (Nonpatent document 6), etc. These genes are considered to be lectin-like proteins having a nucleotide sequence similar to FLO1.
Presently, from the viewpoint of petroleum supply, energy sources are searched at a global level among biological resource (biomass), as an alternative to petroleum. As one of new energies by biomass, there exists bioethanol, which is a biomass fuel. In bioethanol, botanical resources containing a large amount of carbohydrate or starch are utilized. As a method for producing alcohol by microorganism using biomass as raw material, a fermentation production method of ethanol with Saccharomyces cerevisiae comprising saccharizing ground materials of Sago Palm raw wood (Patent Document 3), and a method of producing alcohol for fuel from waste such as garbage by using yeast belonging to Saccharomyces cerevisiae (Patent Document 4) are disclosed. Yeasts having a thermotolerance that can meet biomass treatment and having a high flocculation property similarly as alcohol-producing yeast are awaited to utilize in bioethanol production.
Yeast Kluyveromyces marxianus is a yeast having thermotolerance, and expression of enzyme alcohol dehydrogenase (Adh) associated with the conversion from sugar to ethanol has been confirmed by Lertwattanasakul and Yamada et al. (Nonpatent Document 7). Kluyveromyces marxianus can not only produce ethanol but as it has a high protein productivity it is considered to be very useful in the industrial production. However as a method for transforming Kluyveromyces marxianus is not generally established, studies are not progressing so far. Therefore, Kluyveromyces marxianus transformant strain suitable for industrial production of bioethanol has not at all been reported so far.    Patent Document 1: Japanese Patent No. 3040959    Patent Document 2: Japanese Patent No. 3643404    Patent Document 3: Japanese Laid-Open Patent Application No. 2007-195406    Patent Document 4: Japanese Laid-Open Patent Application No. 2006-325577    Nonpatent Document 1: Biotechnol Lett, 30:97-102, 2008    Nonpatent Document 2: Yeast, 9:423-427, 1993    Nonpatent Document 3: Science, 265:2077-2082, 1994    Nonpatent Document 4: Nature 387:78-81, 1997    Nonpatent Document 5: Proc. Natl. Acad. Sci. U.S.A. 92: 3809-3813, 1995    Nonpatent Document 6: Nature 369: 371-378, 1994    Nonpatent Document 7: Biosci. Biotechnol. Biochem. 71:1170-82, 2007